Advanced micromachining of concave microwells for long term on-chip culture of multicellular tumor spheroids.

A novel approach based on advanced micromachining is demonstrated to fabricate concave microwell arrays for the formation of high quality multicellular tumor spheroids. Microfabricated molds were prepared using a state-of-the-art CNC machining center, containing arrays of 3D convex micropillars with size ranging from 150 μm to 600 μm. Microscopic imaging of the micropillars machined on the mold showed smooth, curved microfeatures of a dramatic 3D shape. Agarose microwells could be easily replicated from the metallic molds. EMT-6 tumor cells seeded in the primary macrowell sedimented efficiently to the bottom of the concave microwells and formed multicellular spheroids within 48 h. Dense and homogeneous multicellular spheroids were obtained after 10 days of culture, confirming the suitability of the proposed approach. To facilitate long term spheroid culture and reliable on-chip drug assay, polydimethylsiloxane microwells were also replicated from the metallic molds. A solvent swelling method was adapted and optimized to Pluronic F127 towards physically entrapping the block copolymer molecules within the polydimethylsiloxane network and in turn to improve long term cell-binding resistance. Homogeneous multicellular spheroids were efficiently formed in the concave microwells and on-chip drug assays could be reliably carried out using curcumin as a model anti-cancer drug. Advanced micromachining provides an excellent technological solution to the fabrication of high quality concave microwells.